Cell Counting and Cell Cycle Analysis of Simple Non-Cultured Endothelial Cell Injection (SNEC-I) Therapy: Characterization for Clinical Translation.

Human corneal endothelial cell therapy has recently emerged as a novel solution to treat corneal endothelial diseases. We previously demonstrated the potential of utilizing non-cultured primary corneal endothelial cells (CEnCs) isolated from donor corneas with low endothelial cell density for simple non-cultured endothelial cell injection (SNEC-I) therapy. This study aimed to develop a robust and semi-automated approach for cell counting, characterize the extent of cellular manipulation, and evaluate the translational workflow. To address this, we evaluated manual and automated cell counting approaches and characterized the extent of manipulation of CEnCs through the analysis of cell cycle status, gene expressions, and transcriptomic profiles with single-cell RNA-sequencing. The translational feasibility and functionality of SNEC-I therapy were examined using an established rabbit model of bullous keratopathy. Manual hemocytometry and automated cell-counters exhibited comparable accuracy and reproducibility. Analysis of cell cycle status, cell cycle genes (n = 11), and transcriptomic profiles revealed close resemblance between the native corneal endothelium and its donor-matched SNEC-I-harvested cells. Successful resolution of bullous keratoplasty in the pre-clinical model supports the feasibility, efficacy, and safety of SNEC-I therapy. In conclusion, SNEC-I therapy serves as an attractive corneal endothelial therapeutic approach (from a regulatory standpoint) in view of the minimal extent of cellular manipulation.